"E1E2" or "P-type" cation-transporting ATPases are widely distributed in the plasma membranes of eukaryotic cells and are also found in bacteria under special conditions of growth. Members of the differ with respect to the cation that is pumped (H+;K+;H+/K+;NA+/K+;Ca2+), but are otherwise structurally and functionally quite similar. Each of the ATPases has a 100kDa catalytic polypeptide with regions of conspicuous sequence homology, and each of the them splits ATP by way of a covalent phosphorylated intermediate. This project will focus on the plasma-membrane (H+)-ATPase of Neurospora as a simple eukaryotic member of the E1E2 group. The gene encoding the ATPase has recently been cloned and sequenced. Three types of experiments are now planned: (1) Group-specific reagents such as N-ethylmaleimide, phenylglyoxal, and p- fluorosulfonylbenzoyl adenosine will be used to identify amino acid residues that play an important role in the reaction cycle of the ATPase, and the residues will be mapped within the 100 kDa polypeptide. (2) Antibodies and proteases will be employed to study the topology of the polypeptide in the membrane. (3) A system will be developed to express site-directed mutants carrying defined changes in the ATPase gene, and the altered enzymes will be characterized with respect to ATP hydrolytic activity and proton pumping. Taken together, the results of these experiments should provide useful information about the molecular mechanism of cation transport.